GSM6730435: C2_003; Mus musculus; RNA-Seq
The left lateral liver lobes were extracted and prepared for FFPE slides. The slides were processed following Manual RNA Slide Preparation Protocol (NanoString, MAN-10115-05 for software v2.3). Briefly, the slides were baked at 60°C for 30min before deparaffinization, rehydration and target retrieval. To expose tissue RNA targets, slides were incubated with 1 ug/mL proteinase K (Ambion, 2546) and the Whole Transcriptome Atlas (WTA) probe reagent at 37°C in a humidified HybEZ II System for 15h. The following day, the optimal diluted solutions of morphology markers, including panCK, CD45 and nuclear staining SYTO13 (Nanostring), were used for distinguishing cell types and degrees of damage. After selecting ROIs, the bound RNA probes were photocleaved and individual segmented areas were collected for sequencing before amplifying Illumina sequencing libraries. AMPure XP beads (Beckman Coulter, A63880) were used to purify PCR reactions at a 1.2× bead-to-sample ratio. DNA high sensitivity bioanalyzer assay (Agilent Technologies) and Qubit dsDNA high sensitivity assay (Thermo Fisher Scientific) were used to evaluate the size and concentration of the sequencing library. Total target counts were calculated from the total sampled areas (μm2) and the sequencing depths were 100 counts / μm2. All sequencing libraries were generated with index uniquely, which were allowed to be pooled together for sequencing. After loaded at a concentration of 365 pM with 5% PhiX, WTA libraries were sequenced with an Illumina NovaSeq 6000 platform under the following parameters: read 1, 27 cycles; read 2, 27 cycles; index 1, 8 cycles; index 2, 8 cycles.